B77-04 Atp-p2x4/p2x7 Signalling Drives Monocyte Cytokine Activation in Progressive Fibrotic Lung Disease and Is Attenuated by Nintedanib

Abstract Rationale Circulating monocytes contribute to inflammation in progressive fibrotic lung disease (IPF/PPF), but upstream drivers of cytokine release remain poorly defined. Extracellular ATP acts as a danger signal via ionotropic P2X receptors. We investigated purinergic receptor expression, ATP-driven cytokine induction, receptor inhibition, and the effect of nintedanib on monocyte activation. Methods Peripheral blood monocytes and plasma were obtained from healthy controls (HC; n = 20), untreated IPF/PPF (n = 20), and IPF/PPF receiving nintedanib (n = 8). P2X receptor expression was quantified by RT-qPCR and Western blot. Plasma IL-1β and TNFR1 were measured by ELISA. Monocytes were stimulated ex vivo with ATP and/or TNF-alpha using time-course analysis, with selective P2X4/P2X7 inhibition. Statistical comparisons used the Mann-Whitney U test. Results Monocytes from IPF/PPF patients demonstrated significantly increased P2X4 and P2X7 expression at mRNA and protein levels compared with HC. Plasma IL-1β (p 0.0001) and TNFR1 (p 0.0001) were elevated in untreated disease. Patients receiving nintedanib exhibited significantly lower IL-1β (p = 0.0008) and TNFR1 (p = 0.002). Baseline monocyte IL-1β production was elevated in IPF/PPF compared with HC. ATP stimulation induced a time-dependent increase in IL-1β protein abundance, with greater magnitude in IPF/PPF monocytes than HC. Combined ATP and TNF-alpha stimulation synergistically amplified IL-1β expression. Selective inhibition of P2X4 and P2X7 attenuated ATP-induced cytokine release. Across endpoints, untreated disease showed heightened baseline cytokine production and exaggerated ATP responsiveness. Conclusions Circulating monocytes in fibrotic lung disease display upregulated P2X4/P2X7 expression and exaggerated ATP-driven cytokine release, amplified by TNF-alpha synergy. Receptor-selective inhibition attenuates this response, indicating a key upstream checkpoint in monocyte activation. Nintedanib reduces circulating cytokines and dampens ATP-induced responses, suggesting an additional immunomodulatory action beyond antifibrotic activity. These data highlight a therapeutically targetable inflammatory axis with relevance to patient treatment and pathway-guided intervention. This abstract is funded by: Bons Secours, Dublin